Nunc labtek 4 well chambered coverglass

A431 cells, seeded onto glass-bottom dishes (Nunc LabTek 4-well chambered coverglass), were first labeled with 50 μg/ml Alexa Fluor 647-dextran (10,000 MW; Thermo Scientific) supplemented with 200 μM oleic acid/BSA in 5% LPDS to label late endosomal organelles and to induce LDs. The cells were then pulse-labeled for 2 h with 50 μg/ml BC LN-LDL in serum-free DMEM. During the last 30 min of LDL labeling, HCS LipidTox Red or HCS LipidTox Deep Red (1:1000; Thermo Scientific) was added to the medium to label LDs. The cells were then washed and chased in serum-free CO₂-independent medium (Gibco) for the indicated times and imaged by live-cell confocal microscopy. Imaging was performed on a Leica TCS SP8 X attached to a motorized DMi8 inverted microscope with ×63 HC PL APO CS2 water objective (1.20 NA). Experiments were performed at 37 °C in CO₂-independent medium (Gibco) supplemented with HCS LipidTox Red/Deep Red (1:1000) in a fully enclosed temperature-controlled environmental chamber. Data were acquired with Leica LAS X (Leica Microsystems). The fraction of BC residing in dextran-positive LEs and LipidTox-positive LDs was quantified from background-subtracted images with ImageJ by using Mander’s overlap coefficient as a measure of colocalization.

The CellEvent Senescence Green Detection Kit (Thermo Fisher Scientific; Waltham, MA, USA) was used. The fluorescein-based probe contains two galactoside moieties, making it a target for cellular β-galactosidase. The enzymatically cleaved product emits fluorescence (absorption/emission maxima = 490/514 nm). In brief, the cells were grown on Nunc Lab-Tek 4-well Chambered Coverglass (Thermo Fisher Scientific; Waltham, MA, USA). After washing with PBS, the cells were fixed with 2% paraformaldehyde and then washed with 1% bovine serum albumin (BSA). Next, the cells were incubated with the diluted CellEvent Senescence Green Probe for 2 h and then washed 3 times with PBS. The samples were observed using a Nikon A1R Si confocal system (Nikon Instruments, Amsterdam, The Netherlands). Nikon NIS Elements AR 4.51 software was applied for image analysis.